Immune reactivity to HER-2/neu protein for diagnosis and treatment of malignancies in which the HER-2/neu oncogene is associated

ABSTRACT

Methods for the detection, monitoring and treatment of malignancies in which the HER-2/neu oncogene is associated are disclosed. Detection of specific T cell activation (e.g., by measuring the proliferation of T cells) in response to in vitro exposure to the HER-2/neu protein, or detection of immunocomplexes formed between the HER-2/neu protein and antibodies in body fluid, allows the diagnosis of the presence of a malignancy in which the HER-2/neu oncogene is associated. The present invention also discloses methods and compositions, including peptides, for treating such malignancies.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/354,533, filed Jul. 15, 1999 now U.S. Pat. No. 6,664,370 and allowed;which application is a continuation of U.S. patent application Ser. No.08/466,680, filed Jun. 6, 1995, which issued as U.S. Pat. No. 6,075,122;which application is a continuation of U.S. patent application Ser. No.08/414,417, filed Mar. 31, 1995 which issued as U.S. Pat. No. 5,801,005;which application is a continuation-in-part of U.S. patent applicationSer. No. 08/106,112, filed Aug. 12, 1993 and abandoned; whichapplication is a continuation-in-part of U.S. patent application Ser.No. 08/033,644, filed Mar. 17, 1993 and abandoned; which applicationsare incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is generally directed toward the detection,monitoring, and treatment of malignancies, in which the HER-2/neuoncogene is associated, through the use of a cancer patient's own immunereactivity to the HER-2/neu protein expressed by the HER-2/neu gene.

2. Description of the Related Art

Despite enormous investments of financial and human resources, cancerremains one of the major causes of death. For example, cancer is theleading cause of death in women between the ages of 35 and 74. Breastcancer is the most common malignancy in women and the incidence fordeveloping breast cancer is on the rise. One in nine women will bediagnosed with the disease. Standard approaches to cure breast cancerhave centered around a combination of surgery, radiation andchemotherapy. These approaches have resulted in some dramatic successesin certain malignancies. However, breast cancer is most often incurable,when diagnosed beyond a certain stage. Alternative approaches to earlydiagnosis and therapy are necessary.

A common characteristic of malignancies is uncontrolled cell growth.Cancer cells appear to have undergone a process of transformation fromthe normal phenotype to a malignant phenotype capable of autonomousgrowth. Amplification and overexpression of somatic cell genes isconsidered to be a common primary event that results in thetransformation of normal cells to malignant cells. The malignantphenotypic characteristics encoded by the oncogenic genes are passed onduring cell division to the progeny of the transformed cells.

Ongoing research involving oncogenes has identified at least fortyoncogenes operative in malignant cells and responsible for, orassociated with, transformation. Oncogenes have been classified intodifferent groups based on the putative function or location of theirgene products (such as the protein expressed by the oncogene).

Oncogenes are believed to be essential for certain aspects of normalcellular physiology. In this regard, the HER-2/neu oncogene is a memberof the tyrosine protein kinase family of oncogenes and shares a highdegree of homology with the epidermal growth factor receptor. HER-2/neupresumably plays a role in cell growth and/or differentiation. HER-2/neuappears to induce malignancies through quantitative mechanisms thatresult from increased or deregulated expression of an essentially normalgene product.

HER-2/neu (p185) is the protein product of the HER-2/neu oncogene. TheHER-2/neu gene is amplified and the HER-2/neu protein is overexpressedin a variety of cancers including breast, ovarian, colon, lung andprostate cancer. HER-2/neu is related to malignant transformation. It isfound in 50%-60% of ductal in situ carcinoma and 20%-40% of all breastcancers, as well as a substantial fraction of adenocarcinomas arising inthe ovaries, prostate, colon and lung. HER-2/neu is intimatelyassociated not only with the malignant phenotype, but also with theaggressiveness of the malignancy, being found in one-fourth of allinvasive breast cancers. HER-2/neu overexpression is correlated with apoor prognosis in both breast and ovarian cancer. HER-2/neu is atransmembrane protein with a relative molecular mass of 185 kd that isapproximately 1255 amino acids (aa) in length. It has an extracellularbinding domain (ECD) of approximately 645 aa, with 40% homology toepidermal growth factor receptor (EGFR), a highly hydrophobictransmembrane anchor domain (TMD), and a carboxyterminal cytoplasmicdomain (CD) of approximately 580 aa with 80% homology to EGFR.

An approach to developing a diagnostic assay for malignancies, in whichthe HER-2/neu oncogene is associated, has been to attempt to quantifythe protein expression product of the HER-2/neu oncogene in tissue orbody fluids. However, there have been problems in the development ofdiagnostic assays based on direct detection of HER-2/neu protein.

Due to the difficulties in the current approaches to diagnosis andtherapy of cancers in which the HER-2/neu oncogene is associated, thereis a need in the art for improved methods and compositions. The presentinvention fills this need, and further provides other relatedadvantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides a variety of methods forthe detection of a malignancy in a warm-blooded animal, wherein aHER-2/neu oncogene is associated with the malignancy. The methods may beused on a one time basis when a malignancy is suspected or on a periodicbasis, e.g., to monitor an individual with an elevated risk of acquiringor reacquiring a malignancy. In one embodiment, the method comprises thesteps of: (a) isolating CD4⁺T cells from a warm-blooded animal; (b)incubating the T cells with HER-2/neu protein; and (c) detecting thepresence or absence of specific activation of the T cells, therebydetermining the presence or absence of the malignancy. In anotherembodiment, the method comprises the steps of: (a) isolating CD8⁺T cellsfrom a warm-blooded animal; (b) incubating the T cells with HER-2/neuprotein; and (c) detecting the presence or absence of specificactivation of the T cells, thereby determining the presence or absenceof the malignancy. In another embodiment, the method comprises the stepsof: (a) contacting a body fluid, suspected of containing antibodiesspecific for HER-2/neu protein, with HER-2/neu protein; (b) incubatingthe body fluid under conditions and for a time sufficient to allowimmunocomplexes to form; and (c) detecting the presence or absence ofimmunocomplexes formed between the HER-2/neu protein and antibodies inthe body fluid specific for the HER-2/neu protein, thereby determiningthe presence or absence of the malignancy.

In another aspect, the present invention provides methods for monitoringthe effectiveness of cancer therapy in a warm-blooded animal with amalignancy, wherein a HER-2/neu oncogene is associated with themalignancy. Uses of such methods include the early detection of relapse.In one embodiment, the method comprises the steps of: (a) contacting afirst body fluid sample, taken from the warm-blooded animal prior toinitiation of therapy, with HER-2/neu protein; (b) incubating the bodyfluid under conditions and for a time sufficient to allowimmunocomplexes to form; (c) detecting immunocomplexes formed betweenthe HER-2/neu protein and antibodies in the body fluid specific for theHER-2/neu protein; (d) repeating steps (a), (b), and (c) on a secondbody fluid sample taken from the animal subsequent to the initiation oftherapy; and (e) comparing the number of immunocomplexes detected in thefirst and second body fluid samples, thereby monitoring theeffectiveness of the therapy in the animal.

The present invention is also directed toward methods for treating amalignancy in a warm-blooded animal, wherein a HER-2/neu oncogene isassociated with the malignancy. In one embodiment, the method comprisesthe steps of: (a) isolating CD4⁺T cells from a warm-blooded animal; (b)incubating the T cells in the presence of HER-2/neu protein, such thatthe T cells proliferate; and (c) administering to the warm-bloodedanimal an effective amount of the proliferated T cells. In anotherembodiment, the method comprises the steps of: (a) isolating CD8⁺T cellsfrom a warm-blooded animal; (b) incubating the T cells in the presenceof HER-2/neu protein, such that the T cells proliferate; and (c)administering to the warm-blooded animal an effective amount of theproliferated T cells. In another embodiment, the method comprises thesteps of: (a) isolating CD4⁺T cells from a warm-blooded animal; (b)incubating the T cells in the presence of HER-2/neu protein, such thatthe T cells proliferate; (c) cloning one or more cells that proliferatedin the presence of HER-2/neu protein; and (d) administering to thewarm-blooded animal an effective amount of the cloned T cells. Inanother embodiment, the method comprises the steps of: (a) isolatingCD8⁺T cells from a warm-blooded animal; (b) incubating the T cells inthe presence of HER-2/neu protein, such that the T cells proliferate;(c) cloning one or more cells that proliferated in the presence ofHER-2/neu protein; and (d) administering to the warm-blooded animal aneffective amount of the cloned T cells. In yet another embodiment, themethod comprises immunizing the animal with a HER-2/neu peptiderecognized by T cells, the peptide not being the extracellular domain ofthe protein expression product of a HER-2/neu oncogene.

Within a related aspect, the present invention provides anti-cancertherapeutic compositions comprising T cells proliferated in the presenceof HER-2/neu protein, in combination with a pharmaceutically acceptablecarrier or diluent. In addition, a variety of peptides designated forCD8⁺T cell responses are provided which include peptides consistingessentially of:

(Seq. ID No. 1) His-Leu-Tyr-Gln-Gly-Cys-Gln-Val-Val; (Seq. ID No. 2)Pro-Leu-Gln-Pro-Glu-Gln-Leu-Gln-Val; (Seq. ID No. 3)Pro-Leu-Thr-Ser-Ile-Ile-Ser-Ala-Val; (Seq. ID No. 4)Ile-Leu-Leu-Val-Val-Val-Leu-Gly-Val; (Seq. ID No. 5)Leu-Leu-Val-Val-Val-Leu-Gly-Val-Val; (Seq. ID No. 6)Arg-Leu-Leu-Gln-Glu-Thr-Glu-Leu-Val; (Seq. ID No. 7)Cys-Leu-Thr-Ser-Thr-Val-Gln-Leu-Val; (Seq. ID No. 8)Asp-Leu-Ala-Ala-Arg-Asn-Val-Leu-Val; (Seq. ID No. 9)Val-Leu-Val-Lys-Ser-Pro-Asn-His-Val; (Seq. ID No. 10)Thr-Leu-Ser-Pro-Gly-Lys-Asn-Gly-Val; (Seq. ID No. 11)Val-Leu-Gly-Val-Val-Phe-Gly-Ile-Leu; (Seq. ID No. 12)Leu-Ile-Lys-Arg-Arg-Gln-Gln-Lys-Ile; (Seq. ID No. 13)Lys-Ile-Pro-Val-Ala-Ile-Lys-Val-Leu; (Seq. ID No. 14)lIe-Leu-Asp-Glu-Ala-Tyr-Val-Met-Ala; (Seq. ID No. 15)Gln-Leu-Met-Pro-Tyr-Gly-Cys-Leu-Leu; (Seq. ID No. 16)Gln-Ile-Ala-Lys-Gly-Met-Ser-Tyr-Leu; (Seq. ID No. 17)Leu-Leu-Asn-Trp-Cys-Met-Gln-Ile-Ala; (Seq. ID No. 18)Arg-Leu-Val-His-Arg-Asp-Leu-Ala-Ala; (Seq. ID No. 19)Asp-Ile-Asp-Glu-Thr-Glu-Tyr-His-Ala; (Seq. ID No. 20)Asp-Leu-Leu-Glu-Lys-Gly-Glu-Arg-Leu; (Seq. ID No. 21)Thr-Ile-Asp-Val-Tyr-Met-Leu-Met-Val; (Seq. ID No. 22)Met-Ile-Met-Val-Lys-Cys-Trp-Met-Ile; (Seq. ID No. 23)Asp-Leu-Val-Asp-Ala-Glu-Glu-Tyr-Leu; (Seq. ID No. 24)Gly-Leu-Glu-Pro-Ser-Glu-Glu-Glu-Ala; or (Seq. ID No. 25)Tyr-Leu-Thr-Pro-Gln-Gly-Gly-Ala-Ala.

Similarly, a variety of peptides designated for CD4+T cell responses areprovided which include peptides consisting essentially of:

His-Leu-Asp-Met-Leu-Arg-His-Leu-Tyr-Gln-Gly-Cys-Gln-Val-Val; (Seq. IDNo. 30) Pro-Leu-Gln-Arg-Leu-Arg-Ile-Val-Arg-Gly-Thr-Gln-Leu-Phe-Glu;(Seq. ID No. 31)Leu-Arg-Ser-Leu-Thr-Glu-Ile-Leu-Lys-Gly-Gly-Val-Leu-Ile-Gln; (Seq. IDNo. 32) Val-Thr-Tyr-Asn-Thr-Asp-Thr-Phe-Glu-Ser-Met-Pro-Asn-Pro-Glu;(Seq. ID No. 33)His-Leu-Arg-Glu-Val-Arg-Ala-Val-Thr-Ser-Ala-Asn-Ile-Gln-Glu; (Seq. IDNo. 34) Val-Arg-Ala-Val-Thr-Ser-Ala-Asn-Ile-Gln-Glu-Phe-Ala-Gly-Cys;(Seq. ID No. 35)Asn-Ile-Gln-Glu-Phe-Ala-Gly-Cys-Lys-Lys-Ile-Phe-Gly-Ser-Leu; (Seq. IDNo. 36) Gln-Val-Phe-Glu-Thr-Leu-Glu-Glu-Ile-Thr-Gly-Tyr-Leu-Tyr-Ile;(Seq. ID No. 37)Gln-Glu-Cys-Val-Glu-Glu-Cys-Arg-Val-Leu-Gln-Gly-Leu-Pro-Arg; (Seq. IDNo. 38) Val-Val-Val-Leu-Gly-Val-Val-Phe-Gly-Ile-Leu-Ile-Lys-Arg-Arg;(Seq. ID No. 39)Lys-Tyr-Thr-Met-Arg-Arg-Leu-Leu-Gln-Glu-Thr-Glu-Leu-Val-Glu; (Seq. IDNo. 40) Gly-Ala-Met-Pro-Asn-Gln-Ala-Gln-Met-Arg-Ile-Leu-Lys-Glu-Thr;(Seq. ID No. 41)Val-Lys-Val-Leu-Gly-Ser-Gly-Ala-Phe-Gly-Thr-Val-Tyr-Lys-Gly; (Seq. IDNo. 42) Ser-Pro-Lys-Ala-Asn-Lys-Glu-Ile-Leu-Asp-Glu-Ala-Tyr-Val-Met;(Seq. ID No. 43)Gly-Val-Gly-Ser-Pro-Tyr-Val-Ser-Arg-Leu-Leu-Gly-Ile-Cys-Leu; (Seq. IDNo. 44) Ser-Arg-Leu-Leu-Gly-Ile-Cys-Leu-Thr-Ser-Thr-Val-Gln-Leu-Val;(Seq. ID No. 45)Gly-Ser-Gln-Asp-Leu-Leu-Asn-Trp-Cys-Met-Gln-Ile-Ala-Lys-Gly; (Seq. IDNo. 46) Val-Lys-Ile-Thr-Asp-Phe-Gly-Leu-Ala-Arg-Leu-Leu-Asp-Ile-Asp;(Seq. ID No. 47)Thr-Val-Trp-Glu-Leu-Met-Thr-Phe-Gly-Ala-Lys-Pro-Tyr-Asp-Gly; (Seq. IDNo. 48) Pro-Ala-Arg-Glu-Ile-Pro-Asp-Leu-Leu-Glu-Lys-Gly-Glu-Arg-Leu;(Seq. ID No. 49)Arg-Phe-Arg-Glu-Leu-Val-Ser-Glu-Phe-Ser-Arg-Met-Ala-Arg-Asp; (Seq. IDNo. 50) Glu-Asp-Asp-Asp-Met-Gly-Asp-Leu-Val-Asp-Ala-Glu-Glu-Tyr-Leu;(Seq. ID No. 51)Gly-Met-Gly-Ala-Ala-Lys-Gly-Leu-Gln-Ser-Leu-Pro-Thr-His-Asp; (Seq. IDNo. 52) Thr-Cys-Ser-Pro-Gln-Pro-Glu-Tyr-Val-Asn-Gln-Pro-Asp-Val-Arg;(Seq. ID No. 53)Thr-Leu-Glu-Arg-Pro-Lys-Thr-Leu-Ser-Pro-Gly-Lys-Asn-Gly-Val; (Seq. IDNo. 54) Gly-Gly-Ala-Val-Glu-Asn-Pro-Glu-Tyr-Leu-Thr-Pro-Gln-Gly-Gly;(Seq. ID No. 55)Asn-Gln-Glu-Val-Thr-Ala-Glu-Asp-Gly-Thr-Gln-Arg-Cys-Glu-Lys; (Seq. IDNo. 56) Gln-Val-Ile-Arg-Gly-Arg-Ile-Leu-His-Asn-Gly-Ala-Tyr-Ser-Leu;(Seq. ID No. 57)Leu-Gln-Val-Phe-Glu-Thr-Leu-Glu-Glu-Ile-Thr-Gly-Tyr-Leu-Tyr; (Seq. IDNo. 58) Ala-Ser-Pro-Leu-Thr-Ser-Ile-Ile-Ser-Ala-Val-Val-Gly-Ile-Leu;(Seq. ID No. 59)Thr-Gln-Arg-Cys-Glu-Lys-Cys-Ser-Lys-Pro-Cys-Ala-Arg-Val-Cys-Tyr-Gly-Leu;(Seq. ID No. 60)Arg-Leu-Arg-Ile-Val-Arg-Gly-Thr-Gln-Leu-Phe-Glu-Asp-Asn-Tyr-Ala-Leu;(Seq. ID No. 61)Lys-Ile-Phe-Gly-Ser-Leu-Ala-Phe-Leu-Pro-Glu-Ser-Phe-Asp-Gly-Asp; (Seq.ID No. 62)Arg-Arg-Leu-Leu-Gln-Glu-Thr-Glu-Leu-Val-Glu-Pro-Leu-Thr-Pro-Ser; or(Seq. ID No. 63)Glu-Leu-Val-Ser-Glu-Phe-Ser-Arg-Met-Ala-Arg-Asp-Pro-Gln. (Seq. ID No.64)

Additional peptides are provided and include a peptide consistingessentially of the amino acid sequence of FIG. 1 from lysine, amino acid676, to valine, amino acid 1255.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that p185^(HER-2/neu) protein (SEQ ID NO: 68) containsmultiple segments with amino acid sequences appropriate for binding toclass II MHC molecules. Each outlined amino acid represents the centerpoint of an 11-mer peptide with alpha-helical periodicity andamphipathicity. Each underlined amino acid segment represents an epitopecorresponding to Rothbard and Taylor motifs.

FIG. 2 graphically illustrates the results of a low frequency eventscreening from a normal individual indicating that a CD4⁺T cell responsecan be detected against p185^(HER-2/neu) and peptides derived from itsamino acid sequence. The graph represents the data from one normalindividual analyzed with the low frequency screening assayed describedfurther below. Positive responses to the intact protein and two peptideswere detected.

FIG. 3 (panels A-D) graphically illustrates that CD4⁺T cells reactive top185^(HER-2/neu) protein and peptides can be detected in high frequencyfrom patients with HER-2/neu positive breast cancer and can also bedetected in some patients with tumors that test negatively forexpression of p185^(HER-2/neu) protein. All four breast cancer patientsrepresented here, patient A (panel A), patient B (panel B), patient C(panel C), and patient D (panel D), were premenopausal women. Patient Ahad a primary tumor that tested negatively for overexpression ofp185^(HER-2/neu). The other three patients had HER-2/neu positivetumors. A proliferation assay was performed using purified peripheralblood mononuclear cells (PBMC) as described below, with eachexperimental group done in 24 well replicate. Two×10⁵ PBMC/well wereincubated with no antigen, tetanus toxoid (5 μg/ml), p185^(HER-2/neu)(50 μg/ml), or HER-2/neu derived peptides (50 μg/ml) as describedfurther below. After 4 days, wells were pulsed with 1 μCi of tritiatedthymidine (³H-TdR) for 6-8 hours and then counted. The data representsthe mean of 24 determinations of the c.p.m. with standard error barsexpressed.

FIG. 4 (panels A and B) graphically illustrates that CD8⁺CTL specificfor HER-2/neu peptides 48-56 and 789-797 can be generated by in vitroimmunization. Three×10⁷ PBMC from a homozygous HLA-A2 normal donor wereincubated with p48-56 or p789-797^(HER-2/neu) peptides at concentrationsof 10 μg/ml. The lymphocytes were tested for lytic activity after 10 invitro sensitizations (IVS). Data is depicted after the tenth IVS withp48-56 (panel A) or with p789-797 (panel B). Target cells consisted of⁵¹Cr-labeled autologous EBV transformed B lymphocytes which had beenincubated with p48-56 or p789-797^(HER-2/neu) or an irrelevant peptidefor 2 hours prior to use. A four hour chromium release assay (CRA) wasperformed. The results represent the percent specific lysis at theindicated effector:target (E:T) ratio. Target controls of ⁵¹Cr-labeledK562 and Daudi cells were also included to evaluate NK and LAK activity.The execution of the CRA is as described. The results represent thepercent specific lysis at the indicated effector:target (E:T) ratio.

FIG. 5 pictorially illustrates that antibodies are detectable againstp185^(HER-2/neu) in the sera of a breast cancer patient. Lane 1represents the immunoblot of p185 using a HER-2/neu positive breastcancer patient's sera (1:1000 dilution) as primary antibody. The blotwas analyzed as described further below. Lane 2 represents the controlstrip from that experiment developed with c-neu antibody.

FIG. 6 pictorially shows that antibodies in the sera of a breast cancerpatient identify the same p185 band as does a known HER-2/neu-specificantibody (“control antibody”). A membrane preparation from NIH3T3 cells(a murine cell line) that had been transfected with HER-2/neu cDNA(“NIH3T3+H2N”) was tested against control antibody (lane A) or patientsera (lane D). Similarly, a membrane preparation from untransfectedcells (“NIH3T3”) was tested against control antibody (lane B) or patientsera (lane C).

FIG. 7 pictorially illustrates that some breast cancer patients haveantibodies directed to both the extracellular and intracellular domainof the HER-2/neu protein. Sera of breast cancer patients is testedagainst the extracellular domain (“ECD protein”) or the intracellulardomain (“ICD protein”), in lanes A and B, respectively.

FIG. 8 graphically illustrates that rats immunized with peptides derivedfrom the intracellular domain (ICD) portion of rat neu protein developantibody responses to neu protein. An ELISA was performed to evaluatepeptide immunized animals for antibody responses to non-transforming ratneu protein. Each sera was analyzed at a 1:25, 1:50, 1:100, and 1:200dilution. The OD value shown is that of the background wells subtractedfrom the wells coated with neu protein. All data shown is at a rat seraconcentration of 1:25. Control sera was derived from an animal immunizedwith adjuvant alone. Antibody responses titered with decreasing serumconcentrations. Results were reproducible in 3 separately run assays.

FIG. 9 graphically illustrates that rats immunized with peptides derivedfrom the extracellular domain (ECD) portion of rat neu protein developantibody responses to neu protein. ELISA evaluation was performed asdescribed in FIG. 8. All data shown is at a rat sera concentration of1:25. Control sera was derived from an animal immunized with adjuvantalone. Antibody responses titered with decreasing serum concentrations.Results were reproducible in 3 separately run assays.

FIG. 10 graphically shows that epitope analysis of ICD antibodyresponses demonstrates dominant B cell epitopes as well as “determinantspreading” between domains. ELISA analysis for peptide epitopes wasperformed. Each animal's sera was evaluated at dilutions of 1:25, 1:50,1:100, and 1:200 for each peptide analyzed. Antibody responses titeredwith decreasing serum concentrations. All data shown is at a rat seraconcentration of 1:50. Control sera analyzed was pooled sera from 5non-immunized animals. Results were reproducible in 3 separately runassays.

FIG. 11 graphically shows that epitope analysis of ECD antibodyresponses demonstrates dominant B cell epitopes. ELISA analysis forpeptide epitopes was performed. Each animal's sera was evaluated atdilutions of 1:25, 1:50, 1:100, and 1:200 for each peptide analyzed.Antibody responses titered with decreasing serum concentrations. Alldata shown is at a rat sera concentration of 1:50. Control sera analyzedwas pooled sera from 5 non-immunized animals. Results were reproduciblein 3 separately run assays.

FIG. 12 pictorially illustrates that antibodies elicited by immunizationto either ICD or ECD peptides are specific for and can immunoprecipitateboth rat neu protein and human HER-2/neu protein. Panel A shows theresults of an immunoprecipitation experiment with immunized rat sera andlysates of DHFRG-8. Each sera was able to immunoprecipitate rat neu fromthe cell lysates. The immunoprecipitates were resolved on a 7.5%SDS-acrylamide gel and transferred to nitrocellulose. The blots wereprobed with primary antibody, c-neu-Ab-3, at a 1:1000 dilution. Controlsera of an animal immunized with the adjuvant alone showed no evidenceof reactivity to rat neu. Panel B depicts the results of animmunoprecipitation experiment with immunized rat sera and lysates ofSKBR3, a source of human neu. Immunoblotting was performed in anidentical manner and all experimental animal sera were able toimmunoprecipitate human neu. The control sera, again, showed no evidenceof reactivity.

FIG. 13 pictorially illustrates that B cell epitopes that are crossreactive between human and rat neu are present in both domains of theprotein. Shown here are the results of Western blot analysis of proteindomain epitope mapping from representative animals in each immunizedgroup. Animal 1.2 was immunized with the ECD group of peptides, animal2.2 with the ICD group of peptides, and the control animal was immunizedwith adjuvant alone. Proteins were electrophoresed. After transfer tonitrocellulose the blots were incubated for 18 hours in rat sera at a1:500 dilution. Antibody responses were detected with a second step goatanti-rat Ig HRP antibody at a dilution of 1:5000. Responses weredetected to both human and rat neu as well as to both human ICD and ECDdomain recombinant proteins. Antibody responses to these proteins couldnot be detected in the control animal which was immunized with adjuvantalone. Although data are shown here for animals 1.2, 2.2, and control,all animals in each group had the same pattern of response.

FIG. 14 graphically illustrates that immunization of rats with ICDpeptides elicits neu peptide-specific T cell responses. 2×10⁵ immunizedspleen cells were incubated with 25 μg/ml of the various peptides. The“Mix” group consisted of 25 μg/ml each of the immunizing peptides. Aproliferation assay was performed. Each experimental group was done in 6well replicates. The data is expressed in terms of a stimulation index(SI) which is the mean of the experimental wells divided by the mean ofthe control (no antigen) wells. Stimulation indices greater than 2 areconsidered to be indicative of a primed response. Animals immunized withadjuvant alone showed no stimulation index greater than 0.9 to any ofthe tested peptides (data not shown).

FIG. 15 graphically illustrates that immunization of rats with ICDpeptides elicits neu protein-specific T cell responses. 1×10⁵ cultured Tcells derived from immunized spleen were incubated with 1×10⁵ syngeneicspleen as APC (antigen presenting cells) and 1 μg/ml of purified rat neuprotein. Each experimental group was done in 6 well replicates. The datais expressed in terms of a stimulation index which is the mean of theexperimental wells divided by the mean of the control (no antigen)wells. Stimulation indices greater than 2 are considered to beindicative of a primed response. Wild type ras protein was theirrelevant protein used in the assay.

FIG. 16 graphically shows that immunization of rats with ECD peptideselicits only weak peptide-specific T cell responses. 2×10⁵ immunizedspleen cells were incubated with 25 μg/ml of the various peptides. The“Mix” group consisted of 25 μg/ml each of the immunizing peptides. Eachexperimental group was done in 6 well replicates. The data is expressedin terms of a stimulation index which is the mean of the experimentalwells divided by the mean of the control (no antigen) wells. Stimulationindices greater than 2 are considered to be indicative of a primedresponse. Animals immunized with adjuvant alone showed no stimulationindex greater than 1.0 to any of the tested peptides (data not shown).

FIG. 17 graphically shows that immunization of rats with ECD peptideselicits weak, but positive, responses to neu protein. 1×10⁵ cultured Tcells derived from immunized spleen or lymph nodes were incubated with1×10⁵ syngeneic spleen as APC and 1 μg/ml of purified rat neu protein.Each experimental group was done in 6 well replicates. The data isexpressed in terms of a stimulation index which is the mean of theexperimental wells divided by the mean of the control (no antigen)wells. Stimulation indices greater than 2 are considered to beindicative of a primed response. Wild type ras protein was theirrelevant protein used in the assay.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms to beused hereinafter.

HER-2/neu Protein—as used herein, refers to the p185 protein (also knownas c-erbB2) and peptides thereof which are recognized by helper T cellsor cytotoxic T cells; and may be naturally derived, syntheticallyproduced, genetically engineered, or a functional equivalent thereof,e.g., where one or more amino acids are replaced by other amino acid(s)or non-amino acid(s) which do not substantially affect function.

Proliferation of T cells—as used herein, includes the multiplication ofT cells as well as the stimulation of T cells leading to multiplication,i.e., the initiation of events leading to mitosis and mitosis itself.Methods for detecting proliferation of T cells are discussed below.

As noted above, the present invention is directed toward methods andcompositions for the diagnosis, monitoring and treatment of malignanciesin a warm-blooded animal, wherein an amplified HER-2/neu gene isassociated with the malignancies. Association of an amplified HER-2/neugene with a malignancy does not require that the protein expressionproduct of the gene be present on the tumor. For example, overexpressionof the protein expression product may be involved with initiation of atumor, but the protein expression may subsequently be lost. An effectiveautochthonous immune response may convert a HER-2/neu positive tumor toHER-2/neu negative, but existent immunity will be present and allowdiagnosis.

More specifically, the disclosure of the present invention, in oneaspect, shows that the protein expression product of the HER-2/neu genecan be recognized by thymus-dependent lymphocytes (hereinafter “Tcells”) and, therefore, the autochthonous immune T cell response can beutilized to diagnose, monitor and treat malignancies in which such aprotein is or has been overexpressed. The disclosure of the presentinvention also shows, in another aspect, that sera of patients with amalignancy, in which an amplified HER-2/neu oncogene is associated,contain antibodies to HER-2/neu protein. The autochthonous antibodyresponse can be used to diagnose, monitor and treat malignancies inwhich such a protein is overexpressed.

It is well known that the two major arms of the immune system are: (1)cell-mediated immunity with immune T cells and (2) humoral immunity withantibodies. Further, the immune system normally functions to recognizeand destroy any foreign or aberrant cells in the body. Since theHER-2/neu protein is expressed by some normal cells, tolerance and/oranergy (i.e., diminished reactivity to a specific antigen) is expected.Thus, it is surprising that, as disclosed within the present invention,both T cell and antibody responses to HER-2/neu are detected.

In general, CD4⁺T cell populations are considered to function ashelpers/inducers through the release of lymphokines when stimulated by aspecific antigen; however, a subset of CD4⁺ cells can act as cytotoxic Tlymphocytes (CTL). Similarly, CD8⁺T cells are considered to function bydirectly lysing antigenic targets; however, under a variety ofcircumstances they can secrete lymphokines to provide helper or DTHfunction. Despite the potential of overlapping function, the phenotypicCD4 and CD8 markers are linked to the recognition of peptides bound toclass II or class I MHC antigens. The recognition of antigen in thecontext of class II or class I MHC mandates that CD4⁺ and CD8⁺T cellsrespond to different antigens or the same antigen presented underdifferent circumstances. The binding of immunogenic peptides to class IIMHC antigens most commonly occurs for antigens ingested by antigenpresenting cells. Therefore, CD4⁺T cells generally recognize antigensthat have been external to the tumor cells. By contrast, under normalcircumstances, binding of peptides to class I MHC occurs only forproteins present in the cytosol and synthesized by the target itself,proteins in the external environment are excluded. An exception to thisis the binding of exogenous peptides with a precise class I bindingmotif which are present outside the cell in high concentration. Thus,CD4⁺ and CD8⁺T cells have broadly different functions and tend torecognize different antigens as a reflection of where the antigensnormally reside.

As disclosed within the present invention, the protein product expressedby the HER-2/neu oncogene is recognized by T cells. Such a proteinexpression product “turns over” within cells, i.e., undergoes a cyclewherein a synthesized protein functions and then eventually is degradedand replaced by a newly synthesized molecule. During the protein lifecycle, peptide fragments from the protein bind to majorhistocompatibility complex (MHC) antigens. By display of a peptide boundto MHC antigen on the cell surface and recognition by host T cells ofthe combination of peptide plus self MHC antigen, a malignant cell willbe immunogenic to T cells. The exquisite specificity of the T cellreceptor enables individual T cells to discriminate between proteinfragments which differ by a single amino acid residue.

During the immune response to a peptide, T cells expressing a T cellreceptor with high affinity binding of the peptide-MHC complex will bindto the peptide-MHC complex and thereby become activated and induced toproliferate. In the first encounter with a peptide, small numbers ofimmune T cells will secrete lymphokines, proliferate and differentiateinto effector and memory T cells. The primary immune response will occurin vivo but has been difficult to detect in vitro. Subsequent encounterwith the same antigen by the memory T cell will lead to a faster andmore intense immune response. The secondary response will occur eitherin vivo or in vitro. The in vitro response is easily gauged by measuringthe degree of proliferation, the degree of cytokine production, or thegeneration of cytolytic activity of the T cell population re-exposed inthe antigen. Substantial proliferation of the T cell population inresponse to a particular antigen is considered to be indicative of priorexposure or priming to the antigen.

Within one aspect of the present invention, a malignancy in which aHER-2/neu oncogene is associated may be detected. Representativeexamples of such malignancies include breast, ovarian, colon, lung andprostate cancers. An immune response to the HER-2/neu protein, oncegenerated, can be long-lived and can be detected long afterimmunization, regardless of whether the protein is present or absent inthe body at the time of testing. In one embodiment, prior exposure of awarm-blooded animal, such as humans, to the HER-2/neu protein can bedetected by examining for the presence or absence of specific activationof CD4⁺ or CD8⁺T cells. More specifically, T cells isolated from anindividual by routine techniques (such as by Ficoll/Hypaque densitygradient centrifugation of peripheral blood lymphocytes) are incubatedwith HER-2/neu protein. For example, T cells may be incubated in vitrofor 2-9 days (typically 4 days) at 37° C. with HER-2/neu protein(typically, 5 μg/ml of whole protein or 25 μg/ml of an appropriatepeptide or graded numbers of cells synthesizing HER-2/neu protein). Itmay be desirable to incubate another aliquot of a T cell sample in theabsence of HER-2/neu protein to serve as a control.

Specific activation of CD4⁺ or CD8⁺T cells may be detected in a varietyof ways. Methods for detecting specific T cell activation includedetecting the proliferation of T cells, the production of cytokines(e.g., lymphokines), or the generation of cytolytic activity (i.e.,generation of cytotoxic T cells specific for HER-2/neu protein). ForCD4⁺T cells, a preferred method for detecting specific T cell activationis the detection of the proliferation of T cells. For CD8⁺T cells, apreferred method for detecting specific T cell activation is thedetection of the generation of cytolytic activity.

Detection of the proliferation of T cells may be accomplished by avariety of known techniques. For example, T cell proliferation can bedetected by measuring the rate of DNA synthesis. T cells which have beenstimulated to proliferate exhibit an increased rate of DNA synthesis. Atypical way to measure the rate of DNA synthesis is, for example, bypulse-labeling cultures of T cells with tritiated thymidine, anucleoside precursor which is incorporated into newly synthesized DNA.The amount of tritiated thymidine incorporated can be determined using aliquid scintillation spectrophotometer. Other ways to detect T cellproliferation include measuring increases in interleukin-2 (IL-2)production, Ca²⁺ flux, or dye uptake, such as3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Alternatively,synthesis of lymphokines (such as interferon-gamma) can be measured orthe relative number of T cells that can respond to intactp185^(HER-2/neu) protein or peptide may be quantified.

Intact p185^(HER-2/neu) protein or peptides thereof which are recognizedby cytotoxic T cells may be used within the present invention. Thepeptides may be naturally derived or produced based upon an identifiedsequence. The peptides for CD8⁺T cell responses are generally 8-10 aminoacids in length and the peptides for CD4⁺T cell responses are longer,e.g., 15-18 amino acids in length. Peptides for CD8⁺T cell responsesvary according to each individual's class I MHC molecules. An example ofpeptides appropriate for CD8⁺T cell responses (elicited by peptidespresented by HLA-A2.1 class I MHC molecules) are peptides which are 8-10amino acids in length and contain a leucine at position 2 and/or aleucine or valine at position 9. Examples of peptides (designated by oneletter abbreviations for amino acids and followed in parentheses bywhich residues of p185 they correspond) suitable within the presentinvention for CD8⁺T cell responses in individuals that are HLA-A2.1include peptides consisting essentially of: HLYQGCQVV (p48-56) (Seq. IDNo. 1); OLFEDNYAL (p106-114) (Seq. ID No. 26); KIFGSLAFL (p369-377)(Seq. ID No. 27); PLQPEQLQV (p391-399) (Seq. ID No. 2); PLTSIISAV(p650-658) (Seq. ID No. 3); ILLVVVLGV (p661-669) (Seq. ID No. 4);LLVVVLGW (p662-670) (Seq. ID No. 5); RLLQETELV (p689-697) (Seq. ID No.6); ILDEAYVMAGV (p767-777) (Seq. ID No. 28); VMAGVGSPYV (p773-782) (Seq.ID No. 29); CLTSTVQLV (p789-797) (Seq. ID No. 7); DLAARNVLV (p845-853)(Seq. ID No. 8); VLVKSPNHV (p851-859) (Seq. ID No. 9); TLSPGKNGV(p1172-1180) (Seq. ID No. 10); VLGVVFGIL (p666-674) (Seq. ID No. 11);LIKRRQQKI (p674-682) (Seq. ID No. 12); KIPVAIKVL (p747-755) (Seq. ID No.13); ILDEAYVMA (p767-775) (Seq. ID No. 14); QLMPYGCLL (p799-807) (Seq.ID No. 15); QIAKGMSYL (p829-836) (Seq. ID No. 16); LLNWCMQIA (p822-830)(Seq. ID No. 17); RLVHRDLAA (p840-848) (Seq. ID No. 18); DIDETEYHA(p871-879) (Seq. ID No. 19); DLLEKGERL (p933-941) (Seq. ID No. 20);TIDVYMLMV (p948-956) (Seq. ID No. 21); MIMVKCWMI (p953-961) (Seq. ID No.22); DLVDAEEYL (p1016-1024) (Seq. ID No. 23); GLEPSEEEA (p1062-1070)(Seq. ID No. 24); or YLTPQGGAA (p1196-1204) (Seq. ID No. 25).

Peptides for CD4⁺T cell responses vary according to each individual'sclass II MHC molecules. Examples of peptides suitable within the presentinvention for CD4⁺T cell responses include peptides consistingessentially of: HLDMLRHLYQGCQVV (p42-56) (Seq. ID No. 30);PLQRLRIVRGTQLFE (p95-109) (Seq. ID No. 31); RLRIVRGTQLFEDNYAL (p98-114)(Seq. ID No. 61); LRSLTEILKGGVLIQ (p142-156) (Seq. ID No. 32);VTYNTDTFESMPNPE (p272-286) (Seq. ID No. 33); NQEVTAEDGTQRCEK (p319-333)(Seq. ID No. 56); TQRCEKCSKPCARVCYGL (p328-345) (Seq. ID No. 60);HLREVRAVTSANIQE (p349-363) (Seq. ID No. 34); VRAVTSANIQEFAGC (p353-367)(Seq. ID No. 35); NIQEFAGCKKIFGSL (p360-374) (Seq. ID No. 36);KIFGSLAFLPESFDGD (p369-384) (Seq. ID No. 62); LQVFETLEEITGYLY (p397-411)(Seq. ID No. 58); QVFETLEEITGYLYI (p398-412) (Seq. ID No. 37);QVIRGRILHNGAYSL (p429-443) (Seq. ID No. 57); QECVEECRVLQGLPR (p538-552)(Seq. ID No. 38); ASPLTSIISAVVGIL (p648-662) (Seq. ID No. 59);VVVLGVVFGILIKRR (p664-678) (Seq. ID No. 39); KYTMRRLLQETELVE (p684-698)(Seq. ID No. 40); RRLLQETELVEPLTPS (p688-703) (Seq. ID No. 63);GAMPNQAQMRILKET (p704-718) (Seq. ID No. 41); VKVLGSGAFGTVYKG (p723-737)(Seq. ID No. 42); SPKANKEILDEAYVM (p760-774) (Seq. ID No. 43);GVGSPYVSRLLGICL (p776-790) (Seq. ID No. 44); SRLLGICLTSTVQLV (p783-797)(Seq. ID No. 45); GSQDLLNWCMQIAKG (p818-832) (Seq. ID No. 46);VKITDFGLARLLDID (p859-873) (Seq. ID No. 47); TVWELMTFGAKPYDG (p911-925)(Seq. ID No. 48); PAREIPDLLEKGERL (p927-941) (Seq. ID No. 49);RFRELVSEFSRMARD (p968-982) (Seq. ID No. 50); ELVSEFSRMARDPQ (p971-984)(Seq. ID No. 64); EDDDMGDLVDAEEYL (p1010-1024) (Seq. ID No. 51);GMGAAKGLQSLPTHD (p1091-1105) (Seq. ID No. 52); TCSPQPEYVNQPDVR(p1132-1146) (Seq. ID No. 53); TLERPKTLSPGKNGV (p1166-1180) (Seq. ID No.54); or GGAVENPEYLTPQGG (p1188-1202) (Seq. ID No. 55).

It will be evident to those of ordinary skill in the art that otherpeptides may be produced for use within the present invention, both forthe HLA-A2.1 class I MHC molecule as well as for the other class I andclass II molecules. A variety of techniques are well known for isolatingor constructing peptides. Suitable peptides are readily identified basedupon the disclosure provided herein. Additional suitable peptidesinclude those which are longer in length. For example, another peptidehas an amino acid sequence corresponding to that disclosed in FIG. 1beginning at about the lysine residue at amino acid position 676 andextending to about the valine residue at amino acid position 1255. Sucha peptide may be extended (e.g., by the addition of one or more aminoacid residues selected, for example, from position 675 to about position646 of FIG. 1) and/or truncated (e.g., by the deletion of one or moreamino acid residues from the carboxyl terminus which is position 1255 ofFIG. 1). Alternatively, suitable peptides may be variations on otherpreferred peptides disclosed herein. For example, variations on thepeptide designated herein as p650-658 include the extension and/ortruncation by the addition or deletion, respectively, of one or moreamino acid residues beginning at either position 650 or position 658 orboth positions. As an example, four amino acids are removed from theamino terminus of p650-658 and four amino acids, such as the fouradjacent to position 658, are added to its carboxyl terminus. Althoughthis particular peptide variation results in a peptide with the samenumber of total amino acids (nine), a peptide variation on a preferredpeptide need not be identical in length. Variations in amino acidsequence that yield peptides having substantially the same desiredbiological activity are within the scope of the present invention.

For therapeutic purposes, CD4⁺ or CD8⁺T cells that proliferate in thepresence of HER-2/neu protein can be expanded in number either in vitroor in vivo. Proliferation of such T cells in vitro may be accomplishedin a variety of ways. For example, the T cells can be re-exposed toHER-2/neu protein. It may be desirable to repeat the exposure of T cellsto the HER-2/neu protein to induce proliferation. It may be furtherdesirable to include T cell growth factors, such as interleukin-2,and/or stimulator cells which synthesize HER-2/neu protein. The additionof stimulator cells is preferred where generating CD8⁺T cell responses.HER-2/neu protein-specific T cells can be grown to large numbers invitro with retention of specificity in response to intermittentrestimulation with the immunizing HER-2/neu protein. Briefly, for theprimary in vitro stimulation (IVS), large numbers of lymphocytes (e.g.,greater than 4×10⁷) are placed in flasks with media containing humanserum. HER-2/neu protein (e.g., peptide at 10 μg/ml) is added directlyas well as 5 μg/ml tetanus toxoid. The flasks are incubated at 37° C.for 7 days. For the second IVS, at the end of the 7 days, T cells areharvested and placed in new flasks with 2-3×10⁷ irradiated peripheralblood mononuclear cells. HER-2/neu protein (e.g., peptide at 10 μg/ml isadded directly). The flasks are incubated at 37° C. for 7 days. On day 2and day 4 after the second IVS, 2-5 units of interleukin-2 (IL-2) isadded. For the third IVS, the T cells are placed in wells (e.g., 24 wellplates). The T cells are stimulated with the individual's own EBVtransformed B cells coated with the peptide. IL-2 is added on days 2 and4 of each cycle. As soon as the cells are shown to be specific cytotoxicT cells, they are changed to a 10 day stimulation cycle with higher IL-2(20 units) on days 2, 4 and 6 to expand them.

Alternatively, one or more T cells that proliferate in the presence ofHER-2/neu protein can be expanded in number by cloning. Methods forcloning cells are well known in the art. For example, T cell lines maybe established in vitro and cloned by limiting dilution. Responder Tcells are purified from the peripheral blood of sensitized patients bydensity gradient centrifugation and sheep red cell rosetting andestablished in culture by stimulating with the nominal antigen in thepresence of irradiated autologous filler cells. In order to generateCD4⁺T cell lines, HER-2/neu protein is used as the antigenic stimulusand autologous peripheral blood lymphocytes (PBL) or lymphoblastoid celllines (LCL) immortalized by infection with Epstein Barr virus are usedas antigen presenting cells. In order to generate CD8⁺T cell lines,autologous antigen-presenting cells transfected with an expressionvector which produces relevant HER-2/neu protein may be used asstimulator cells. Established T cell lines are cloned 2-4 days followingantigen stimulation by plating stimulated T cells at a frequency of 0.5cells per well in 96-well flat-bottom plates with 1×10⁶ irradiated PBLor LCL cells and recombinant interleukin-2 (rIL2) (50 U/ml). Wells withestablished clonal growth are identified at approximately 2-3 weeksafter initial plating and restimulated with appropriate antigen in thepresence of autologous antigen-presenting cells, then subsequentlyexpanded by the addition of low doses of rIL2(10 U/ml) 2-3 daysfollowing antigen stimulation. T cell clones are maintained in 24-wellplates by periodic restimulation with antigen and rIL2 approximatelyevery two weeks.

Regardless of how an individual's T cells are proliferated in vitro, theT cells may be administered to the individual as an anti-cancercomposition in an amount effective for therapeutic attack against atumor. Thus, a patient's own T cells (autochthonous T cells) can be usedas reagents to mediate specific tumor therapy. Typically, about 1×10⁹ to1×10¹¹ T cells/M² will be administered intravenously or intracavitary,e.g., in pleural or peritoneal cavities, or in the bed of a resectedtumor. It will be evident to those skilled in the art that the numberand frequency of administration will be dependent upon the response ofthe patient. Pharmaceutically suitable carriers or diluents for T cellsinclude physiological saline or sera. It will be recognized by oneskilled in the art that the composition should be prepared in sterileform.

T cells may also be proliferated in vivo. For example, immunization ofan individual with a HER-2/neu peptide (i.e., as a vaccine) can inducecontinued expansion in the number of T cells necessary for therapeuticattack against a tumor in which the HER-2/neu oncogene is associated.Typically, about 0.01 μg/kg to about 100 mg/kg body weight will beadministered by the intradermal, subcutaneous or intravenous route. Apreferred dosage is about 1 μg/kg to about 1 mg/kg, with about 5 μg/kgto about 200 μg/kg particularly preferred. It will be evident to thoseskilled in the art that the number and frequency of administration willbe dependent upon the response of the patient. It may be desirable toadminister the HER-2/neu peptide repetitively. It will be evident tothose skilled in this art that more than one HER-2/neu peptide may beadministered, either simultaneously or sequentially. For example, acombination of about 8-15 peptides may be used for immunization.Preferred peptides for immunization are those that include all or aportion of the amino acid sequence shown in FIG. 1 beginning at aboutthe lysine residue at amino acid position 676 and extending to about thevaline residue at amino acid position 1255. One or more peptides fromother portions of the amino acid sequence shown in FIG. 1 may be addedto one or more of the preferred peptides. Neither intactp185^(HER-2/neu) protein nor a peptide having the amino acid sequence ofits entire extracellular domain (i.e., a peptide having an amino acidsequence of the entire amino acid sequence shown in FIG. 1 up to aminoacid position 650, plus or minus about one to five positions, and withor without the first 21 amino acid positions) are used alone forimmunization.

In addition to the HER-2/neu peptide (which functions as an antigen), itmay be desirable to include other components in the vaccine, such as avehicle for antigen delivery and immunostimulatory substances designedto enhance the protein's immunogenicity. Examples of vehicles forantigen delivery include aluminum salts, water-in-oil emulsions,biodegradable oil vehicles, oil-in-water emulsions, biodegradablemicrocapsules, and liposomes. Examples of immunostimulatory substances(adjuvants) include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP),lipopoly-saccharides (LPS), glucan, IL-12, GM-CSF, gamma interferon andIL-15. It will be evident to those skilled in this art that a HER-2/neupeptide may be prepared synthetically or that a portion of the protein(naturally-derived or synthetic) may be used. When a peptide is usedwithout additional sequences, it may be desirable to couple the peptidehapten to a carrier substance, such as keyhole limpet hemocyanin.

The present invention also discloses that HER-2/neu protein, in additionto being immunogenic to T cells, appears to stimulate B-cells to produceantibodies capable of recognizing HER-2/neu protein. Detection of suchantibodies provides another way to diagnose a malignancy in which aHER-2/neu oncogene is associated with the malignancy. Antibodiesspecific (i.e., which exhibit a binding affinity of about 10⁷liters/mole or better) for HER-2/neu protein may be found in a varietyof body fluids including sera and ascites. Briefly, a body fluid sampleis isolated from a warm-blooded animal, such as a human, for whom it isdesired to determine whether antibodies specific for HER-2/neu arepresent. The body fluid is incubated with HER-2/neu protein underconditions and for a time sufficient to permit immunocomplexes to formbetween the protein and antibodies specific for the protein. Forexample, a body fluid and HER-2/neu protein may be incubated at 4° C.for 24-48 hours. Following the incubation, the reaction mixture istested for the presence of immunocomplexes. Detection of one or moreimmunocomplexes formed between HER-2/neu protein and antibodies specificfor HER-2/neu protein may be accomplished by a variety of knowntechniques, such as radioimmunoassays (RIA) and enzyme linkedimmunosorbent assays (ELISA).

Suitable immunoassays include the double monoclonal antibody sandwichimmunoassay technique of David et al. (U.S. Pat. No. 4,376,110);monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkhamand Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone,Edinburgh, 1970); the “western blot” method of Gordon et al. (U.S. Pat.No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J.Biol. Chem. 255:4980-4983, 1980); enzyme-linked immunosorbent assays asdescribed by, for example, Raines and Ross (J. Biol. Chem.257:5154-5160, 1982); immunocytochemical techniques, including the useof fluorochromes (Brooks et al., Clin. Exp. Immunol. 39: 477, 1980); andneutralization of activity [Bowen-Pope et al., Proc. Natl. Acad. Sci.USA 81:2396-2400 (1984)], all of which are hereby incorporated byreference. In addition to the immunoassays described above, a number ofother immunoassays are available, including those described in U.S. Pat.Nos. 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; and 4,098,876, all of which are herein incorporated byreference.

For detection purposes, HER-2/neu protein (“antigen”) may either belabeled or unlabeled. When unlabeled, the antigen find use inagglutination assays. In addition, unlabeled antigen can be used incombination with labeled molecules that are reactive withimmunocomplexes, or in combination with labeled antibodies (secondantibodies) that are reactive with the antibody directed againstHER-2/neu protein, such as antibodies specific for immunoglobulin.Alternatively, the antigen can be directly labeled. Where it is labeled,the reporter group can include radioisotopes, fluorophores, enzymes,luminescers, or dye particles. These and other labels are well known inthe art and are described, for example, in the following U.S. Pat. Nos.3,766,162; 3,791,932; 3,817,837; 3,996,345; and 4,233,402.

Typically in an ELISA assay, antigen is adsorbed to the surface of amicrotiter well. Residual protein-binding sites on the surface are thenblocked with an appropriate agent, such as bovine serum albumin (BSA),heat-inactivated normal goat serum (NGS), or BLOTTO (buffered solutionof nonfat dry milk which also contains a preservative, salts, and anantifoaming agent). The well is then incubated with a sample suspectedof containing specific antibody. The sample can be applied neat, or,more often, it can be diluted, usually in a buffered solution whichcontains a small amount (0.1%-5.0% by weight) of protein, such as BSA,NGS, or BLOTTO. After incubating for a sufficient length of time toallow specific binding to occur, the well is washed to remove unboundprotein and then incubated with an anti-species specific immunoglobulinantibody labeled with a reporter group. The reporter group can be chosenfrom a variety of enzymes, including horseradish peroxidase,beta-galactosidase, alkaline phosphatase, and glucose oxidase.Sufficient time is allowed for specific binding to occur, then the wellis again washed to remove unbound conjugate, and the substrate for theenzyme is added. Color is allowed to develop and the optical density ofthe contents of the well is determined visually or instrumentally.

In one preferred embodiment of this aspect of the present invention, areporter group is bound to HER-2/neu protein. The step of detectingimmunocomplexes involves removing substantially any unbound HER-2/neuprotein and then detecting the presence or absence of the reportergroup.

In another preferred embodiment, a reporter group is bound to a secondantibody capable of binding to the antibodies specific for HER-2/neuprotein. The step of detecting immunocomplexes involves (a) removingsubstantially any unbound antibody, (b) adding the second antibody, (c)removing substantially any unbound second antibody and then (d)detecting the presence or absence of the reporter group. Where theantibody specific for HER-2/neu protein is derived from a human, thesecond antibody is an anti-human antibody.

In a third preferred embodiment for detecting immunocomplexes, areporter group is bound to a molecule capable of binding to theimmunocomplexes. The step of detecting involves (a) adding the molecule,(b) removing substantially any unbound molecule, and then (c) detectingthe presence or absence of the reporter group. An example of a moleculecapable of binding to the immunocomplexes is protein A.

It will be evident to one skilled in the art that a variety of methodsfor detecting the immunocomplexes may be employed within the presentinvention. Reporter groups suitable for use in any of the methodsinclude radioisotopes, fluorophores, enzymes, luminescers, and dyeparticles.

In a related aspect of the present invention, detection ofimmunocomplexes formed between HER-2/neu protein and antibodies in bodyfluid which are specific for HER-2/neu protein may be used to monitorthe effectiveness of cancer therapy for a malignancy in which theHER-2/neu oncogene is associated. Samples of body fluid taken from anindividual prior to and subsequent to initiation of therapy may beanalyzed for the immunocomplexes by the methodologies described above.Briefly, the number of immunocomplexes detected in both samples arecompared. A substantial change in the number of immunocomplexes in thesecond sample (post-therapy initiation) relative to the first sample(pre-therapy) reflects successful therapy.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 CD4+T Cells Responsive to p185(HER-2/neu) Protein andPeptides can be Detected in Higher Frequency in Patients with BreastCancer than Normal Individuals

A. p185(HER-2/neu) Protein Contains Multiple Segments with Amino AcidSequences Appropriate for Binding to Class II MHC Molecules

Soluble proteins are classically processed in the class II MHC pathway.p185^(HER-2/neu) protein is a transmembrane protein present at the cellsurface. When overexpressed, it has been found to be soluble and in theextracellular environment both in vitro and in vivo. In vitro studies ofhuman breast cancer cell lines found the extracellular domain ofp185^(HER-2/neu) in culture media of rapidly growing cells (Alper etal., Cell Growth and Differentiation 1:591-599, 1990; Zabrecky et al.,J. Biol. Chem. 266:1716-1720, 1991). In vivo studies identifiedcirculating portions of the protein in the sera of patients with breastcancer (Leitzel et al., J. Clin. Oncol. 10:1436-1443, 1992; Mori et al.,Jpn. J. Cancer Res. 81:489-494, 1990).

Peptide segments of the parental HER-2/neu protein with a motif withtheoretical potential to bind to class II MHC molecules were identifiedherein. Locating potential T cell epitopes was aided by computeranalysis. A protein sequence analysis package, T Sites, thatincorporates several computer algorithms designed to distinguishpotential sites for T cell recognition was used (Feller and de la Cruz,Nature 349:720-721, 1991). Two searching algorithms were used: (1) theAMPHI algorithm described by Margalit (Feller and de la Cruz, Nature349:720-721, 1991; Margalit et al., J. Immunol. 138:2213-2229, 1987)identified epitope motifs according to alpha-helical periodicity andamphipathicity; (2) the Rothbard and Taylor algorithm identified epitopemotifs according to charge and polarity pattern (Rothbard and Taylor,EMBO 7:93-100, 1988). Segments with both motifs are most appropriate forbinding to class II MHC molecules, with the caveat that each particularMHC molecule has a particular binding motif. Using this analysis, morethan 40 potential T cell epitopes in the HER-2/neu protein correspondingto the AMPHI and the Rothbard motifs that would have the potential forbinding to class II MHC molecules were identified (FIG. 1).

Peptides, each 15 amino acids in length, that encompass both the AMPHIand Rothbard motifs were constructed. The optimal peptide length forclass II MHC binding depends upon the particular MHC molecule and may beshorter than 15 amino acids. However, class II MHC responses toexogenous peptides allow for endocytosis and intracellular processing oflonger peptides. One of the synthetic peptides (p42-56), HLDMLRHLYQGCQVV(Seq. ID No. 30), is located in the extracellular domain and has 33%homology to epidermal growth factor receptor (EGFR). Two other syntheticpeptides, SRLLGICLTSTVQLV (p783-797) (Seq. ID No. 45) andTLERPKTLSPGKNGV (p1166-1180) (Seq. ID No. 54) are both located in theintracellular domain and have 87% and 7% homology to EGFR respectively.The peptides as well as partially purified whole protein(p185^(HER-2/neu)) were used in subsequent defined experiments to detectCD4+T cell proliferation responses (Section C below).

B. p185(HER-2/neu) Protein can be Obtained and Purified from the HumanBreast Adenocarcinoma Cell Line SKBR3

Purified p185 for T cell proliferation studies and antibody detectionstudies was obtained from the cell line SKBR3. SKBR3 has been reportedon extensively in the literature as a commonly used standard cell linewith increased HER-2/neu gene copy number and HER-2/neu proteinoverexpression. In one study, SKBR3 cells were found to contain a meanHER-2/neu oncogene copy number of 43 copies/cell compared with 2.5copies/cell for MCF-7, a breast cancer cell line considered to be astandard cell line without HER-2/neu gene amplification (Kallionieme etal., Proc. Nat. Acad. Sci. USA 89:5321-5325, 1992). SKBR3 is reported tobe one of the highest known expressors of p185^(HER-2/neu) protein byimmunohistochemistry, 4+ compared to 1+ in MCF-7 (Kerns et al., J.Histochem. & Cytochem. 38:1823-1830, 1990). The same HER-2/neu bands asdescribed in the literature were validated in the present experiments byWestern analysis. Bands detected included p185, p105 (extracellulardomain), and several smaller bands that presumably represent fragmentsof phosphorylated protein (Alper et al., Cell Growth and Differentiation1:591-599, 1990; Zabrecky et al., J. Biol. Chem. 266:1716-1720, 1991;Stern et al., Mol. Cell. Biol. 8:3969-3973, 1988).

The antibodies used for detecting the HER-2/neu protein immunoblottingwere commercially prepared by Oncogene Science (Manhasset, N.Y.). Theantibody most commonly used in the present experiments was c-neu Ab-3;derived by immunization of BALB/c mice with a peptide sequence,TAENPEYLGLDVPV (Seq. ID No. 65), from the carboxyl domain of human c-neugene product, and fusion of mouse splenocytes with SP2/0 myeloma cells.A second antibody, c-neu AB-1, gave very faint bands when compared withc-neu Ab-3. This antibody was a polyclonal rabbit affinity purifiedantibody against the peptide sequence, LARLLDIDETEYAD (Seq. ID No. 66),from the kinase domain of the human c-neu gene product.

Transmembrane p185^(HER-2/neu) protein was purified from the cellmembrane fraction of SKBR3 by modifications of described methods forother membrane-associated proteins (Dhut et al, Leukemia 4:745-750,1990; Mietzner et al., J. Exp. Med. 165:1041-1057, 1987). Three×10⁶SKBR3 cells were harvested and suspended in phosphate buffered saline(PBS) with the following protease inhibitors; 1 mM PMSF, 1 mMbenzamidine, 5 μg/ml aprotinin. All procedures were done on ice or at 4°C. The cells were then disrupted by sonication at 75W for a total of 1minute using a high intensity sonifier equipped with a microtip (BransonInstruments, Inc., Stamford, Conn.). The resulting suspension was thencentrifuged for 1 hour at 35,000 rpm to sediment membranes fromcytosolic fraction. The membrane pellet was washed in ice cold PBS withprotease inhibitors and the cycle of sonication/centrifugation wasrepeated twice. All cytosolic (supernatant) and membranous fractionswere tested for the presence of p185^(HER-2/neu) by Western analysis.The protein was noted to be strongly concentrated in the membranefraction.

Protein concentration of one of these enriched membrane pellets wasdetermined to be 2625 μg/ml (Protein BioRad assay). p185^(HER-2/neu) isan estimated 8% of membrane protein in SKBR3 (Leitzel et al., J. Clin.Oncol. 10:1436-1443, 1992); therefore, an estimated 210 μg ofp185^(HER-2/neu) were present in the membrane pellet from 3×10⁶ SKBR3cells.

If desired, the membrane preparation may be further enriched forp185^(HER-2/neu), e.g., by immunoprecipitation. Briefly, 1 μg of c-neu 3antibody and 15 μl protein A agarose were added to the sonicatedmembrane pellet. The mixture was incubated at 4° C. on a rocker for 24hours. The immunoprecipitate was collected by centrifugation in amicro-centrifuge at 2500 rpm for 15 minutes at 4° C., and the resultingpellet was washed several times with PBS, 1% Trition X-100, 0.5% sodiumdeoxycholate, and 0.1% sodium dodecyl sulfate. Silver stain and Westernanalysis showed increased concentration of p185^(HER-2/neu) protein anddecreases in extraneous membrane proteins when compared to membraneenriched pellets alone.

C. CD4+T Cells Reactive to p185(HER-2/neu) Protein can be Elicited fromPBL of Normal Individuals by Using an Assay Designed for Detecting LowFrequency Lymphocyte Precursors

Three assays were used for the detection of CD4⁺ responses: a standardproliferation assay, a screening method for low frequency events, and alimiting dilution assay (LDA). Conventional proliferative assays arecapable of readily detecting primed responses. The proliferativeresponse stimulation index provides a rough correlation with precursorfrequency of antigen-reactive T cells. Any specific proliferativeresponse detected from PBL is considered to be a primed response.

To provide a more quantitative interpretation of CD4⁺T cell responses,the assay system developed for detecting low lymphocyte precursorfrequency responses (described below) is used. This assay is simple andcost-effective. In circumstances in which more precision is needed, theprecursor frequency is validated by limiting dilution assays (Bishop andOrosz, Transplantation 47:671-677, 1989).

Responses greater than detected in normal individuals are defined as aprimed response and imply existent immunity. Low responses, detectableonly by LDA conditions are considered to be unprimed responses. Anabsent response by LDA or a response lower than that defined by thenormal population analysis is considered to be tolerance/anergy.

In general, primed CD4⁺T cell responses can be detected in conventionalproliferative assays, whereas unprimed responses are not detectable inthe same assays. Detection of small numbers of unprimed T cells islimited by confounding background thymidine uptake including theautologous mixed lymphocyte response (AMLR) to self MHC antigen plusresponses to processed self serum proteins and exogenously added serumproteins.

To elicit and detect unprimed T cells, an assay system for low frequencyresponses based on Poisson sampling statistics was used (In: Pinnacles,Chiron Corporation, 1:1-2, 1991). This type of analysis appliesspecifically to low frequency events in that, if the precursor frequencyis less than the number of cells in one replicate culture, manyreplicates are required to detect a statistically significant number ofpositives. Theoretically, the analysis will correct for autologousresponses by setting up a known positive control (such as PHA or tetanustoxoid) and known negative control (no antigen) and evaluating all datapoints from lowest to highest irrespective of the experimental group towhich they belong. A cutoff value is calculated based on the equationcutoff=M+(F+SD), where M=arithmetic mean, F=3.29, a factor from tablesof standardized normal distribution chosen so not more than 0.1% of the“true negatives” of a normally distributed background will be above thecutoff, and SD=standard deviation. In this screening assay, wells abovethe cutoff are considered true positives that potentially contain alymphocyte that is specifically proliferating to the antigen ofinterest. Although estimations of lymphocyte precursor frequency ispossible using this method, precise determination requires formal LDAanalysis.

Analysis of PBL from normal individuals for HER-2/neu peptide andprotein-specific T cells revealed the presence of a low level frequencyof proliferative responses. A representative assay is described in FIG.2. Seven normal subjects were analyzed, 4 males and 3 females. Of theseven individuals evaluated, 57% had a response to whole protein and 29%had a response to at least one individual peptide. The two individualsthat responded to peptide also had responses to parental protein. Threemales and one female had detectable responses to the whole protein. Twomales responded to one of the four peptides. Similar methods can be usedto elicit HER-2/neu reactive T cells from patients with HER-2/neupositive malignancies, but no prior priming in vivo. Alternatively, themethods can be used to assess the efficacy of priming to HER-2/neu invivo and the procurement of immune T cells to be expanded for therapy.

D. CD4+T Cells Reactive to p185(HER-2/neu) and Peptide can be Detectedin the Peripheral Blood of Patients with HER-2/neu Positive BreastCancer in Levels Consistent with a Primed Response

Four breast cancer patients with known HER-2/neu tumor status have beenevaluated in a standard proliferation assay. Three patients had tumorswhich overexpressed the HER-2/neu protein. Proliferation to antigen wasconsistent with a primed response (FIG. 3) (i.e., proliferation wasdetectable in a standard proliferation assay with a Stimulation Index(S.I.) greater than 2). One patient was HER-2/neu negative and had aresponse towards intact HER-2/neu, but no response to HER-2/neu-derivedpeptides. The patients tested and chosen had different stages of diseaseand were in different stages of treatment. Five normal individuals'responses were analyzed in the same fashion, and none had an S.I.greater than 2 to any HER-2/neu protein or peptide (Table 1).

TABLE 1 HER-2/neu TetanusT p783– p1166– Patient Status oxoid p185 p42–56797 1180 (a) Breast Cancer patients A Negative 52 13 2 2 2 B Positive 36<2 26 19 2 C Positive 7 4 <2 <2 <2 D Positive 10 4 4 5 <2 TetanusT p783–p1166– Normal oxoid p185 p42–56 797 1180 (b) Normal Individuals 1 6 2 2<2 <2 2 7 <2 2 <2 <2 3 7 <2 <2 <2 <2 4 10 2 ND ND <2 5 11 2 ND ND 2

Example 2

CD8⁺CTL Specific for HER-2/neu Peptides can be Generated from PBL ofNormal Individuals by Primary in Vitro Immunization to SyntheticPeptides Derived from the Normal Amino Acid Sequence of p185(HER-2/neu)Protein

A. p185(HER-2/neu) Protein Contains Multiple Segments with an Amino AcidSequence Motif Appropriate for Binding the Class I MHC Molecule HLA-A2.1

CD8⁺T cells recognize peptide bound to class I MHC molecules. Ingeneral, peptide determinants are derived from endogenously synthesizedproteins. The rules which determine the ability of a protein to beprocessed and complexed with class I MHC molecules are not completelyunderstood. Recently, however, it has been determined that peptidesbinding to particular MHC molecules share discernible sequence motifs(Falk et al., Nature 351:290-296, 1991). A peptide motif for binding inthe groove of HLA-A2.1 has been defined by Edman degradation of peptidesstripped from HLA-A2.1 molecules of a cultured cell line (Table 2, fromFalk et al., supra). The method identified the typical or averageHLA-A2.1 binding peptide as being 9 amino acids in length with dominantanchor residues occurring at positions 2 (L) and 9 (V). Commonlyoccurring strong binding residues have been identified at positions 2(M), 4 (E,K), 6 (V), and 8 (K). The identified motif represents theaverage of many binding peptides.

TABLE 2 The HLA-A2.1 Restricted Motif Point Amino Acid Position Assign-1 2 3 4 5 6 7 8 9 ment Dominant Binding L V +3 Anchor Residue StrongBinding M E V K +2 Residue K Weak Binding I A G I I A E L +1 Residue L YP K L Y S F F D Y T H K P T N M M G Y S V H

The derived peptide motif as currently defined is not particularlystringent. Some HLA-A2.1 binding peptides do not contain both dominantanchor residues and the amino acids flanking the dominant anchorresidues play major roles in allowing or disallowing binding. Not everypeptide with the current described binding motif will bind, and somepeptides without the motif will bind. However, the current motif isvalid enough to allow identification of some peptides capable ofbinding.

According to the current motif, the p185^(HER-2/neu) protein contains asubstantial number of peptides with amino acid sequences possiblyappropriate for binding to the class I MHC antigen HLA-A2.1. Evaluationof the 1255 aa structure of p185^(HER-2/neu) revealed at least 19peptide segments of 9 aa in length that contained at least one of thedominant anchor residues. Of note, the current HLA-A2.1 motif places 6amino acids between the dominant anchor amino acids at residues 2 and 9.Recent studies show that alterations in secondary structure of peptidescan sometimes allow for additional intervening residues, and thus longerbinding peptides. In the present experiment, 9-mer peptides wereevaluated. The 10 peptides with both dominant residues were considered.The arbitrary scoring system awarded +3 for a dominant anchor residue,+2 for a strong binding residue, and +1 for a weak binding residue.Emphasis was placed on presence or absence of dominant anchor residuesas they appear to be of prime importance for peptide binding to HLA-A2(Parker et al., J. Immunol. 148:3580-3587, 1992). Four peptides weresynthesized (Table 3). One is located in the extracellular domain of theprotein and three are located in the intracellular domain. Homology toEGFR ranges from 11% to 89% (Bargmann et al., Nature 319:226-230, 1986).

TABLE 3 p185^(HER-2/neu) Peptides Constructed for Binding in HLA-A2.1Motif p185^(HER-2/neu) Amino Acid Position Homology Peptides 1 2 3 4 5 67 8 9 Score Location to EGFR p 48–56^(HER-2/neu) H L Y Q G C Q V V 8*Extracellular 33% (Seq. ID No. 1) p 789–797^(HER-2/neu) C L T S T V Q LV 9* Intracellular 89% (Seq. ID No. 7) p 851–859^(HER-2/neu) V L V K S PN H V 9* Intracellular 78% (Seq. ID No. 9) p 1172–1180^(HER-2/neu) T L SP G K N G V 9* Intracellular 11% (Seq. ID No. 10) *Peptide contains bothdominant anchor residuesB. Four of Four Peptides with a Motif Theoretically Appropriate forBinding to HLA-A2.1 can be Shown to Actually Bind to HLA-A2.1 in a ClassI MHC Molecule Stabilization Assay

Having identified and synthesized peptides with a theoretical likelihoodof binding to HLA-A2.1, the constructed peptides were evaluated as towhether in fact they could bind, the sine quo non of cytotoxic Tlymphocytes (CTL) generation. Of the four peptides constructed, allcould be shown to bind to HLA-A2 in an assay utilizing the mutant cellline T2. T2 is a human T-B cell hybrid that has a large homozygousdeletion within the MHC gene region (Riberdy and Cresswell, J. Immunol.148:2586-2590, 1992; Trousdale et al., Nature 348:741-744, 1990; Spieset al., Nature 348:744-747, 1990). The use of T2 to determine HLA-A2.1binding peptides has been well defined. T2 does not appropriatelyprocess endogenous antigen for presentation with class I MHC molecules.Consequently, cell surface expression of class I MHC molecules ismarkedly reduced. However, provision of exogenous peptides which bind toand stabilize class I MHC in the presence of B2 microglobulin results inincreased levels of class I at cell surface which can be easily detectedby immunofluorescent staining. T2 without exogenous peptide has lowexpression of HLA-A2 (30%-50%). When incubated with peptides able tobind A2, the level of class I MHC stabilizes on the cell surface and canbe measured by immunofluorescent staining. Thus, the T2 line fails topresent internal proteins in the class I pathway, but can bind exogenouspeptides, providing that the exogenous peptides have the appropriateHLA-A2.1 binding motif.

In this experiment, 1×10⁶ T2 cells were incubated with individualpeptides at a concentration of 25 μg/ml for 18 hours at 37° C. Bindingof peptides to HLA-A2 was determined by immunofluorescent staining witha mouse monoclonal HLA-A2 antibody followed by rabbit antimouse IgG-FITCconjugate. The peptides which bound HLA-A2 increased class I surfaceexpression to 60%-85% (10-15 percentage points over baseline).

C. CD8⁺CTL Specific for HER-2/neu p48-56 and p789-797 can be Generatedby Primary in Vitro Immunization

In general, detection of T cell responses in vitro implies prior priminghas occurred in vivo. It has been difficult and rare to generate CTL invitro from unprimed populations.

Conditions for detecting immunity to standard recall antigens were usedand no peptide-specific CTL could be detected. A set of conditions werederived which have allowed priming to 4 of 4 of the binding peptidestested to date. The conditions were derived by empiric experimentationbut are consistent with the current paradigm. Conditions include: (1)large numbers of T cells; (2) a concurrent stimulated primed CD4⁺T cellresponse; (3) IL-2 added late to culture in very small amounts; and (4)multiple restimulations.

Initial experiments examined response to p48-56 which is normallypresent in the extracellular domain and p789-797 which is normallypresent in the intracellular domain, both of which were found to bind toHLA-A2.1. Four of four peptides with a motif theoretically appropriatefor binding to HLA-A2.1 are shown to actually bind to HLA-A2.1 in aclass I MHC molecule stabilization assay (Table 4). T2 cells wereincubated for 18 hours with the depicted synthetic p185^(HER-2/neu)peptides. Cells were then washed and stained with antihuman HLA-A2antibody (3%), a second step FITC-conjugated antibody (3%) was thenadded. The % increase of class I on cell surface as measured byincreased fluorescent intensity of cells incubated with peptide comparedto cells incubated in medium alone is indicated.

TABLE 4 p185^(HER-2/neu) % Increase of class I Peptides stabilization onT2 p 48–56 20% p 789–797 20% p 851–859 12% p 1172–1180 10%

After leukapheresis of a normal homozygous HLA-A2 individual, bulkcultures of lymphocytes (3×10⁷) were incubated with peptide in aconcentration of 10 μg peptide/ml. An individual homozygous for HLA-A2.1was used on the presumption that a double dose of the MHC/peptidecomplex would allow more effective priming. Large numbers of lymphocyteswere used to overcome the presumed low frequency of precursors.Generation of CD8⁺CTL responses has long been known to requireconcurrent stimulation of CD4⁺T cell responses to providehelp/amplification. Both peptides used were chosen for class I MHCbinding, and presumably could not stimulate CD4⁺ helper T cells. Toprovide T cell help, low concentrations (5 μg/ml) of tetanus toxoid wereadded to culture along with peptide. So as not to overwhelm or dominatethe culture with the tetanus toxoid response, titrations of tetanustoxoid had previously been assessed in a standard proliferation assaywith the donor's lymphocytes and the concentration of tetanus toxoidthat provided the lowest detectable stimulation index was used.

Low doses of IL-2 added late to culture were used to maintain lymphocyteproliferation. Within the present disclosure, standard conditions forexpanding in vivo primed CTL following secondary sensitization in vitrousually have included IL-2 at 5-10 U/ml on day 2 of stimulation. Underprimary in vitro immunization conditions, similar concentrations of IL-2induced expansion of nonspecifically lytic NK and T cells, presumablydue to the predominance of NK cells and AMLR responsive cells relativeto peptide-specific CTL. For in vitro priming, the T cell culturereceived no IL-2 for the first 10 days, with only 1 unit/ml administeredon day +2 after the second IVS. Thereafter, IL-2 at 2 U/ml could beadministered on day +2 and day +4 of the 7 day stimulation cycle. Tcells were stimulated with peptide on irradiated PBL as APC every 7days. Evaluation for specific lytic function was performed after thefourth IVS and revealed specific lytic activity but substantialnon-specific lytic NK and T cell activity. Routine ⁵¹Cr release assayperformed after the tenth IVS (FIG. 4) revealed greater than 50% lysisfor both bulk T cell lines. Lysis against control targets of K562 andDaudi was less than 2%.

Example 3 Antibodies Directed against HER-2/neu Protein can be Detectedin the Sera of Patients with Breast Cancer

A. Antibodies Directed Against p185(HER-2/neu) Protein andp105(HER-2/neu) Extracellular Domain were Detected in the Sera of SomeBreast Cancer Patients

The sera of 20 patients with breast cancer were analyzed. The 20patients were participants from the Fred Hutchinson Cancer ResearchCenter, Division of Epidemiology WISH study. The patient populationconsisted of women recently diagnosed with breast cancer, generally lessthan 3 months from surgery. Their age was less than 55 and theirHER-2/neu tumor status was unknown. Anti-p185 antibody was found in 55%of the group as evidenced by bands corresponding to the positivecontrol.

Antibody analysis was based on a modification of standard Westernblotting techniques (Laemmli, Nature 227:680-685, 1970; Burnett, Anal.Biochem. 112:195-203, 1981). A 7.5% SDS polyacrylamide gel was pouredwith a single 12 cm long comb in the stacking gel to create a “trough.”Two immunoprecipitated SKBR3 membrane preparations, described above,were dissolved in loading buffer and layered across the trough. The gelwas then run in standard fashion resulting in a band of equallydistributed proteins across the gel. The protein was transferred tonitrocellulose (Amersham Hybond) for subsequent immunoblotting anddevelopment by chemiluminescence methods (Amersham ECL). Once proteintransfer was complete, the nitrocellulose was cut lengthwise into 25equal strips and placed in a 25 well incubation tray. The nitrocellulosestrips were then blocked with Tris buffered saline and 1% bovine serumalbumin (TBS BSA) for 1 hour. This allows for analysis of 23 patientswith 2 control strips. Patient sera is used as primary antibody, andafter blocking, the strips are incubated for 24 hours at 4° C. with seradiluted 1:200 and 1:400 in TBS BSA. The second antibody is a goatantihuman HRP conjugate which will interact with the chemiluminescentdeveloping reagent (Amersham ECL) resulting in light emission which canbe photographed. A control strip is developed with c-neu Ab3 antibodypreviously described in a similar fashion with this assay both IgA andIgG antibody specific for p185 were detected. Patient sera identifiedthe same p185 band (FIG. 5) as did the known HER-2/neu-specificantibody, providing evidence that some patients have existent antibodyimmunity to HER-2/neu.

To validate these responses patient sera was tested against a murinecell line (NIH 3T3) that had been transfected with HER-2/neu cDNA. As anegative control, untransfected cells were used. Membrane preparationswere prepared from the two cell lines and patient sera was used asprimary antibody as previously described. The patient sera identifiedthe same p185 band as did the known HER-2/neu-specific antibody. Thatband was present in the cells that contained HER-2/neu, but undetectablein the cells that did not contain HER-2/neu (FIG. 6).

Recombinant proteins of the extracellular and intracellular domainportions of HER-2/neu were obtained. The extracellular protein (110 kD)and intracellular protein (75 kD) were resolved on a 7.5% SDS-PAG geland incubated with patient sera as primary antibody as previouslydescribed. The sera identified both proteins proving that some patientshave antibodies directed to both the extracellular and intracellulardomain of the HER-2/neu protein (FIG. 7).

B. Seven Normal Individuals Showed No Evidence of Antibody to HER-2/neuProtein

In studies to determine the extent to which detection of antibody toHER-2/neu is specific for malignancy, sera from seven normal individualswas obtained and analyzed in identical fashion as described above. Therewas no evidence of antibodies directed toward any HER-2/neu protein.

C. The Sera of Three Patients with Known HER-2/neu Positive TumorsContained Antibodies Against p185 and p105

Sera from breast cancer patients whose HER-2/neu tumor status is knownwas collected and analyzed to determine the extent to which antibody toHER-2/neu correlates with the presence of HER-2/neu-positive tumors.Three patients with overexpression of p185^(HER-2/neu) protein in theirprimary tumor were analyzed. Antibodies against p185 were detected inall three. The antibody detected in our studies was IgG. Immunoglobulinclass switch from IgM to IgG or IgA require T cell help often directedagainst different epitopes on the same protein molecule.

Example 4 Peptide Based Vaccines Elicit Immunity to HER-2/neu

A. Materials and Methods

1. Animals

Rats used in this study were Fischer strain 344 (CDF (F-344)/CrlBR)(Charles River Laboratories, Portage Mich.). Animals were maintained atthe University of Washington Animal facilities under specific pathogenfree conditions and routinely used for experimental studies between 3and 4 months of age.

2. Antigens

Nine peptides were constructed, derived from the amino acid sequence ofthe rat neu protein. The peptides, 15-18 amino acids in length, werehighly homologous to the human HER-2/neu peptide sequence. Thesepeptides were chosen based on an increased probability of interactionwith human Class II MHC molecules. This theoretical potential wasevaluated by the use of a protein sequence analysis package, TSites,that incorporates several computer algorithms designed to distinguishpotential sites for T cell recognition (Feller and de la Cruz, Nature349:720-721, 1991). Several peptides identified from the rat sequencewere predicted to have potential for class II interaction with bothhuman and murine MHC. Nine peptides were chosen for immunization of therats (Table 5). Eight of the nine were in areas of 100% homology withhuman neu. The remaining peptide had greater than 80% homology withhuman neu (Yamamoto et al., Nature 319:230-234, 1986). The peptides weresynthesized and purified by H. Zabrowski (University of Washington,Seattle, Wash.), then dissolved in phosphate-buffered saline (PBS), pH7.4, to give 2 mg/ml stock solutions. Prior to aliquoting, peptides weresterile filtered, then stored at −70° C.

TABLE 5 Peptides from the Rat neu Protein for Immunization Rat ProteinHomology to Sequence Amino Acids Domain Human neu p 45–59HLDMLRHLYQGCQVV ECD 100% (Seq. ID No. 30) p 98–112 PLQRLRIVRGTQLFE ECD100% (Seq. ID No. 31) p 323–337 NQEVTAEDGTQRCEK ECD 100% (Seq. ID No.56) p 332–349 TQRCEKCSKPCARVCYGL ECD 100% (Seq. ID No. 60) p 433–447RIIRGRILHDGAYSL ECD  80% (Seq. ID No. 67) p 781–795 GVGSPYVSRLLGICL ICD100% (Seq. ID No. 44) p 788–802 SRLLGICLTSTVQLV ICD 100% (Seq. ID No.45) p 932–946 PAREIPDLLEKGERL ICD 100% (Seq. ID No. 49) p 1171–1185TLERPKTLSPGKNGV ICD 100% (Seq. ID No. 54) ECD = extracellular domain ICD= intracellular domain

3. Immunization

One group of rats was immunized with a mixture of extracellular domain(ECD) peptides and one group with a mixture of intracellular domain(ICD) peptides. The final group received adjuvant alone. Peptides wereadministered at a final concentration of 100 μg each in a total volumeof 200 μl. The animals underwent 3 immunizations each 14-16 days apartwith either CFA or IFA as adjuvant (Sigma ImmunoChemicals, St. Louis,Mo.). 16 days after the third immunization sera was obtained forassessment of immune response.

4. Cell Lines

Two cell lines were used as a source of neu proteins. SKBR3, a humanbreast cancer cell line that is a marked overexpressor of HER-2/neu(American Type Culture Collection, Rockville, Md.), was maintained inculture in 10% fetal bovine serum (FBS) (Gemini Bioproducts, Inc.,Calabasas, Calif.) and RPMI. DHFR-G8, an NIH/3T3 cell line cotransfectedwith cneu-p and pSV2-DHFR (American Type Culture Collection, Rockville,Md.), was used as a source of non-transforming rat neu protein (Bernardset al., Proc. Natl. Acad. Sci. USA 84:6854-6858, 1987). This cell linewas maintained in 10% FBS and Dulbecco's modified Eagle's medium with4.5 g/L glucose. DHFR-G8 cells were passaged through the same mediumsupplemented with 0.3 μM methotrexate at every third passage to maintainthe neu transfectant.

5. Preparation of Cell Lysates

Lysates of both SKBR3 and DHFR-G8 were prepared and used as a source ofprotein for both ELISA and immunoprecipitation studies. Briefly, a lysisbuffer consisting of tris base, sodium chloride and Triton-X (1%) pH 7.5was prepared. Protease inhibitors were added; aprotinin (1 μg/ml),benzamidine (1 mM) and PMSF (1 mM). 1 ml of the lysis buffer was used tosuspend 10⁷ cells. The cells were vortexed for 15 seconds every 10minutes for an hour until disrupted. All procedures were performed onice in a 4° C. cold room. After disruption the cells were microfuged at4° C. for 20 minutes. Supernatant was removed from cell debris andstored in small aliquots at −70° C. until used. Presence of human andrat neu in the lysates was documented by Western blot analysis.

6. ELISA for Rat Antibody Responses

96 well Immulon 4 plates (Baxter SP, Redmond, Wash.: DynatechLaboratories) were incubated overnight at 4° C. with an IgG2a murinemonoclonal antibody directed against rat neu (kindly provided by Dr. M.Green) at a concentration of 10 μg antibody per ml. After incubation,all wells were blocked with PBS and 1% bovine serum albumin (BSA) (SigmaChemical Co., St. Louis, Mo.), 100 μl/well for 4 hours at roomtemperature. The plate was washed with PBS/0.5% Tween and protein wasadded. Rows of wells were coated with alternating PBS/1% BSA and DHFR-G8lysate (10⁸ cells/20 ml PBS), 50 μl/well, overnight at 4° C. Afterwashing, the plate was incubated with rat sera at the followingdilutions: 1:25, 1:50, 1:100, 1:200. The sera was diluted in PBS/1%BSA/1% FBS/25 μg/ml mouse IgG/0.01% NaN₃ and then serially into PBS/1%BSA. 50 μl of diluted sera was added/well and incubated 1 hour at roomtemperature. Sheep anti-rat Ig horseradish peroxidase (HRP) was added tothe wells at a 1:7,500 dilution in PBS/1% BSA and incubated for 45minutes at room temperature (Amersham Co., Arlington Heights, Ill.).Isotype assays were performed similarly with rabbit anti-rat IgG andsheep anti-rat IgM HRP antibodies as the second step antibody at aconcentration of 1:5000 (Serotec Ltd., Oxford, England). Control wellsconsisting of varying dilutions of c-neu-Ab-1, a rabbit polyclonalantibody directed against the kinase portion of human neu which also hasreactivity to rat neu (Oncogene Science, Uniondale, N.Y.), were used asa positive control. These wells received a second step antibody ofgoat-anti rabbit HRP at a 1:5000 dilution (Amersham Co.). Following thefinal wash, TMB (Kirkegaard and Perry Laboratories, Gaithersburg, Md.)developing reagent was added. Color reaction was read at an opticaldensity of 640 nm until the positive control wells reached 0.3 OD. Thereaction was stopped with 1N HCl and the optical density was read at 450nm. The OD of each serum dilution was calculated as the OD of the neucoated wells minus the OD of the PBS/1% BSA coated wells. A pool of 5normal rat sera was run on each plate as a negative control.

7. Immunoprecipitation

Experimental rat sera was used to immunoprecipitate human neu from theSKBR3 cell line and rat neu from the DHFR-G8 cell line. A commerciallyprepared IgG1 mouse monoclonal antibody, c-neu-Ab-3, which cross reactswith both human and rat neu, was used as the positive control antibodyin the immunoprecipitation (Oncogene Science). Sera from 5 pooled normalrats and 2 rats immunized with adjuvant alone and no peptide antigenswere used as 2 negative controls. 1 ml of DHFR-G8 or SKBR3 lysate wasincubated with 75 μl of rat sera or 10 μl (1 μg) of neu specificmonoclonal antibody and 15 μl of protein A+G (Oncogene Science). Thesolution was rocked gently overnight at 4° C. After this incubation, theagarose was pelleted and washed twice in a tris HCl/EDTA buffer (1M TrisHCl pH 7.5, 0.25 M EDTA, and 5M NaCl), then twice in the same bufferwith NP-40 added to a 0.5% concentration. The immunoprecipitates wereanalyzed by Western blot as described above using c-neu-Ab-1 (OncogeneScience) as the primary antibody. This antibody is a neu specificpolyclonal rabbit antibody which cross reacts with both human and ratneu.

8. ELISA for Peptide Epitope Analysis

96 well Immulon 4 plates (Dynatech Laboratories) were incubatedovernight at 4° C. with peptides at a concentration of 10 μg/welldiluted in PBS alternating with rows of PBS/1% BSA. After incubation,all wells were blocked with PBS/1% BSA, 100 μl/well for 4 hours at roomtemperature. The plate was washed with PBS/0.5% Tween. After washing,the plate was incubated with rat sera at the following dilutions: 1:50and 1:100. The sera was diluted in PBS/1% BSA/1% FBS/25 μg/ml mouseIgG/0.01% NaN₃ and then serially into PBS/1% BSA. 50 μl of diluted serawas added/well and incubated 1 hour at room temperature. Sheep anti-ratHRP was added to the wells at a 1:7,500 dilution in PBS/1% BSA andincubated for 45 minutes at room temperature. Following the final wash,the TMB developing reagent was added. Color reaction was read at anoptical density of 640 nm until the reading on the most reactive wellreached 0.3 OD. The reaction was stopped with 1N HCl and the opticaldensity was read at 450 nm. The OD of each serum dilution was calculatedas the OD of the peptide coated wells minus the OD of the PBS/1% BSAcoated wells. A pool of 5 normal rat sera was run with each peptide atthe same dilutions as the experimental sera as a negative control.

9. Western Blot Analysis for Rat Antibody Responses

Immunoprecipitates of SKBR3 and DHFR-G8 were used as a source of humanand rat neu proteins in the Western assays. Recombinant human ECD andICD (kindly provided by Drs. B. Groner and N. Lydon) were used toevaluate antibody responses to the neu domains. 7.5% polyacrylamide gelswere electrophoresed in the Pharmacia Phast System (Pharmacia LKBBiotechnology AB, Uppsala, Sweden). After transfer to nitrocellulose(Hybond-C, Amersham Co.) the neu proteins were identified by immunoblotin a similar manner. All control blots were developed by using the IgG1mouse monoclonal primary antibody, c-neu-Ab-3 (Oncogene Science). Thisantibody cross reacts with both rat and human neu. The primary antibodywas used in a 1:1000 dilution with tris-buffered saline/1% BSA/0.1%Nonidet P-40. A polyclonal rabbit antimouse HRP-conjugated secondantibody (Amersham Co.) was used in a 1:10,000 dilution. The blot wasthen developed using a chemiluminescent reaction (Amersham ECL).Identically run experimental blots were analyzed with rat sera asprimary antibody. The sera were used in a 1:500 dilution withtris-buffered saline/1% BSA/0.1% Nonidet P-40 in an overnight incubationwith the blot a 4° C. Secondary antibody, goat-anti rat HRP conjugate(Amersham Co.) was used at a 1:5000 dilution. The blots were developedwith ECL detection reagents and exposed to Hyperfilm-ECL (Amersham Co.).The film was developed and examined for reaction to human and rat neu aswell as the ICD and ECD domains of the protein. Sera from 5 poolednormal rats and 2 rats immunized with adjuvant alone and no peptideantigens were used as 2 negative controls.

10. T Cell Proliferation Assays

For analysis of neu peptide specific responses: Fresh spleen or lymphnode cells were harvested by mechanical disruption and passage throughwire mesh and washed. 2×10⁵ spleen cells/well and 1×10⁵ lymph nodecells/well were plated into 96-well round bottom microtiter plates(Corning, Corning, N.Y.) with 6 replicates per experimental group. Themedia used consisted of EHAA 120 (Biofluids) with L-glutamine,penicillin/streptomycin, 2-mercaptoethanol, and 5% FBS. Cells wereincubated with 25 μg/ml of the various peptides. The group incubatedwith the peptide mix received 25 μg of each of the peptides. After 4days, wells were pulsed with 1 μCi of [³H]thymidine for 6-8 hours andcounted. Data is expressed as a stimulation index (SI) which is definedas the mean of the experimental wells divided by the mean of the controlwells (no antigen). For analysis of neu protein specific responses:Spleen or lymph node cells were cultured for 3 in vitro stimulations. Atthe time of analysis 1×10⁵ cultured spleen or lymph node T cells wereplated into 96 well microtiter plates as described above. Cells wereincubated with 1 μg/ml immunoaffinity column purified rat neu (fromDHFR-G8 cells as the source of rat neu). After 4 days, wells were pulsedwith 1 μCi of [³H]thymidine for 6-8 hours and counted. Data is expressedas a stimulation index which is defined as the mean of the experimentalwells divided by the mean of the control wells (no antigen).

11. Rat T Cell Culture

Spleen and lymph nodes from immunized rats were harvested into singlecell suspensions. PBMC were isolated by Ficoll/Hypaque density gradientcentrifugation (Histopaque-1083, Sigma Diagnostics, St. Louis, Mo.).Cells were washed and resuspended in bulk culture of 3×10⁷ cells in 6well plates. The media used consisted of EHAA 120 (Biofluids) withL-glutamine, penicillin/streptomycin, 2-mercaptoethanol, and 10% FBS. Amix of the immunizing peptides were added directly to culture at aconcentration of 10 μg/ml of each peptide. The cultures wererestimulated on the peptide mix every 14 days with syngeneic spleen thathad been preincubated with the peptide mix for 2 hours, irradiated to1000 rads, and then washed. Stimulator to effector ratio was 1:1 in eachculture. After the second week in culture, media was supplemented with50% Con A conditioned media. At the end of 3 in vitro stimulations,cells were >98% CD3+.

B. Rats Immunized with Peptides Derived from the ICD Portion of Rat NeuProtein Develop Antibody Responses to Neu Protein

Rats were immunized with mixtures of either 4 ICD peptides or 5 ECDpeptides. Following the third immunization, serum and T cells fromimmunized rats were assessed for immunity to neu peptides and protein.Initial experiments assessed rats immunized with ICD peptides forantibody responses to whole neu protein. Serum antibody responses wereanalyzed by ELISA (FIG. 8). The results demonstrate that immunization toICD peptides elicited antibody to whole neu protein. Sera was analyzedat 1:25, 1:50, 1:100, and 1:200 dilution. Results at the 1:25 dilutionare depicted (FIG. 8). Neu specific antibody responses titered rapidlyand at a 1:200 dilution the experimental sera demonstrated the samelevel of response as control. Isotype analysis revealed that theantibody responses were predominantly IgG (data not shown).

C. Rats Immunized with Peptides Derived from the ECD Portion of Rat NeuProtein Develop Antibody Responses to Neu Protein

Immunizations with ECD peptides were performed in an identical fashionas with ICD peptides. ELISA performed on sera from rats immunized withECD peptides revealed the generation of antibody responses to whole neuprotein (FIG. 9). The responses were equivalent to responses elicited byimmunization with ICD peptides. These responses were predominantly ofthe IgG subtype (data not shown).

D. Epitope Analysis of ICD Antibody Responses Demonstrates Dominant BCell Epitopes as well as “Determinant Spreading” Between Domains

Mixtures of peptides had been used above for immunization. To determinewhich peptides in the mixture were the predominant B cell epitopes, serafrom rats immunized with ICD peptides was analyzed by ELISA forresponses to individual peptides. Responses to both ICD and ECD peptideswere evaluated with the presumption that responses to the ECD peptideswould be non-existent. Results (FIG. 10) revealed different responses ineach rat. All rats had marked antibody responses to the overlapping p781and p788 ICD peptides, although the relative levels of responses variedbetween animals. Responses to p932 and p1171 were observed, but wererelatively weak. Surprisingly, rats immunized to the mixture of ICDpeptides displayed significant antibody responses to ECD peptides.Responses in individual rats varied. Rat 2.2 had substantial responsesto all five ECD peptides evaluated. Rats 2.1 and 2.3 had weakerresponses. Thus, immunization to ICD peptides elicited antibodyresponses to ICD peptides as well as “determinant spreading” with thegeneration of antibody responses to the ECD portion of the molecule.Rats immunized with adjuvant alone did not develop T cell responses toany tested peptide.

E. Epitope Analysis of ECD Antibody Responses Demonstrates Dominant BCell Epitopes

Determination of the dominant B cell epitopes in ECD peptide immunizedanimals was performed in an identical fashion. Again, the relativeresponses to individual peptides differed between each animal. Ratsimmunized with ECD peptides developed substantial responses to p45,p332, and p433 and minimal responses to p98 and p323 (FIG. 11). Thedominant epitope was p45 in rats 1.1 and 1.3, but was p433 in rat 1.2.As with immunization to ECD peptides, determinant spreading wasobserved. All rats developed antibody to p788 in the ICD and rats 1.1and 1.2 responded to p1171. The magnitude and extent of “determinantspreading” appeared to be less in the animals immunized with the ECDpeptides than those immunized with the ICD peptides. However, only alimited number of potential epitopes were examined.

F. Antibodies Elicited by Immunization to either ICD or ECD Peptides areSpecific for and can Immunoprecipitate Both Rat Neu Protein and HumanHER-2/neu Protein

The above experiments showed that immunization to neu peptides couldelicit antibody responses to whole rat protein and peptides, asdetermined by ELISA. Verification of the antibody responses to proteinobserved by ELISA was performed by assessing the ability of immune serato immunoprecipitate rat neu protein from lysates of DHFRG-8, an NIH-3T3cell line transfected with non-transforming rat neu. Results showed thatsera from rats immunized with either ECD or ICD peptides couldimmunoprecipitate rat neu (FIG. 12, Panel A).

The immunizing rat neu peptides were homologous with the human HER-2/neuprotein sequence. Thus, the anti-peptide antibodies elicited should bereactive to both rat and human peptides. To determine whether theantibodies elicited were also specific for human HER-2/neu protein,experiments evaluated the ability of sera from peptide immunized rats toimmunoprecipitate HER-2/neu from lysates of SKBR3, a human breast cancercell line that overexpresses HER-2/neu. Sera from all rats immunizedwith ICD or ECD peptides could immunoprecipitate HER-2/neu protein whilethe control sera did not (FIG. 12, Panel B).

G. B cell Epitopes that are Cross Reactive Between Human and Rat Neu arePresent in Both Domains of the Protein

Antibody elicited by immunization to ICD and ECD peptidesimmunoprecipitated both rat and human neu protein. To further evaluatethe protein domains recognized, sera from rats immunized with ICD andECD peptides was evaluated by Western analysis for reactivity againsthuman recombinant ECD and ICD as well as whole human and rat neuimmunoprecipitated protein. Sera from animals immunized with either ICDor ECD peptides recognized both domains and whole protein from bothspecies (FIG. 13). Control animals had no evidence of antibodiesdirected against either domain. These results verify not only thephenomenon of “determinant spreading” suggested in the peptide epitopeanalysis, but also demonstrate human and rat cross reactive epitopes inboth domains.

H. Immunization of Rats With ICD Peptides Elicits Neu Peptide-Specific TCell Responses

The above-detected antibody responses were IgG implying that T cell helpwas present and operative in immunoglobulin class switch. Spleen andlymph nodes cells were evaluated for proliferative responses to theimmunizing peptides. Proliferative T cell responses to the immunizingpeptides were observed, but the relative responses between individualrats were varied (FIG. 14). A stimulation index of >2 was arbitrarilychosen as the cut off of significance. Rat 2.1 did not have anyproliferative response greater than SI of 2 to the mixture of immunizingICD peptides or to individual peptides. Rats 2.2 and 2.3 had SI>2 to themixture of ICD peptides with the dominant response to p1171 in bothrats.

I. Immunization of Rats With ICD Peptides Elicits Neu Protein-Specific TCell Responses

Peptide specific T cell lines were derived by repeated in vitrostimulation of spleen cells from peptide immunized mice by a mixture ofthe immunizing peptides. After 40 days the cultured cells were greaterthan 98% CD3+. The cultured T cells from 2 of the 3 immunized ratsdemonstrated substantial responses to protein with SIs of 9 and 16 (FIG.15). The SI from the third rat was >2. No responses to control proteinwere observed.

J. Immunization of Rats With ECD Peptides Elicits Only WeakPeptide-Specific T Cell Responses

A similar analysis was performed with T cells derived from animalsimmunized with the ECD peptides. Unlike the responses observed from theanimals immunized with the mixture of ICD peptides, animals immunizedwith ECD peptides exhibited only weak proliferative responses to themixture of ECD peptides as well as to individual peptides (FIG. 16).Only one of three rats displayed SI of 2.0 or greater to peptides.

K. Immunization of Rats with ECD Peptides Elicits Weak, but PositiveResponses to Neu Protein

Both splenic and lymph node T cells derived from ECD peptide immunizedrats were analyzed for responses to rat neu protein (FIG. 17). Splenic Tcells exhibited low level responses, whereas responses were greater forlymph node derived T cells. Proliferative responses were not the samefor all animals tested. The maximum SI for spleen derived T cell lineswas 2.1, whereas the maximum SI for lymph node derived T cells was 3.

From the foregoing, it will be evident that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. An isolated peptide consisting of the amino acid sequence ofHis-Leu-Tyr-Gln-Gly-Cys-Gln-Val-Val (Seq. ID No. 1).
 2. An isolatedpeptide consisting of the amino acid sequence ofPro-Leu-Gln-Pro-Glu-Gln-Leu-Gln-Val (Seq. ID No. 2).
 3. An isolatedpeptide consisting of the amino acid sequence ofCys-Leu-Thr-Ser-Thr-Val-Gln-Leu-Val (Seq. ID No. 7).
 4. An isolatedpeptide consisting of the amino acid sequence ofVal-Leu-Val-Lys-Ser-Pro-Asn-His-Val (Seq. ID No. 9).
 5. An isolatedpeptide consisting of the amino acid sequence ofGln-Leu-Met-Pro-Tyr-Gly-Cys-Leu-Leu (Seq. ID No. 15).